Fungus gnat pollination of the inconspicuous orchid Conchidium muscicola in the rainy season of southern Yunnan, China

Abstract

The Orchidaceae, comprising nearly 30,000 species, ranks among the most species-rich families of angiosperms, a diversity widely considered to be driven by floral variation and diverse pollination mechanisms (Tremblay et al., 2005). Most orchids exhibit highly specific pollination systems, with their characteristically low fruit sets often linked to pollinator limitation (Ackerman, 1989; Tremblay et al., 2005). Despite more than a century of pollination research since Darwin's pioneering work, over 90% of orchid species remain unstudied (Ackerman et al., 2023). This knowledge gap is particularly pronounced for taxa with inconspicuous floral traits that evade visual detection, constraining our understanding of orchid adaptive radiation and pollination syndrome evolution. Dipterans represent the second most important group of pollinators after hymenopterans (Larson et al., 2001) and contribute significantly to orchid pollination systems (Jiang et al., 2020). Among Diptera, micro-dipterans such as fungus gnats (Mycetophilidae, Sciaridae, Cecidomyiidae, Keroplatidae, Bolitophilidae, Diadocidiidae, and Ditomyiidae) are particularly noteworthy, characterized by small body sizes ( 0.05; one-way ANOVA), demonstrating that C. muscicola requires insect pollination but maintains self-compatibility. Four-year observations (2019–2022) revealed consistently high natural fruit sets ranging from 33.06% to 61.72% across both populations (Figures 1d and 2a; Appendix S1: Table S2), with the LBZ population exhibiting significantly higher reproductive success than JMM in three of the four years (Figure 2a). Furthermore, we identified a positive correlation between floral display size (flowers per plant) and fruit set in both populations (Figure 2b), supporting the hypothesis that increased floral displays enhance pollinator attraction and consequently improve orchid reproductive success (Suetsugu et al., 2015). Overall, our findings demonstrate that Sciaridae (Bradysia) fungus gnats serve as effective and specialized pollinators of C. muscicola. Fungus gnat pollination remains exceptionally rare in orchids, with most documented cases involving Mycetophilidae species. To our knowledge, this study reports only the third known instance of Sciaridae functioning as orchid pollinators worldwide, while additionally revealing novel pollination strategies that extend beyond the sexual deception mechanisms previously described in Pterostylis and Lepanthes (Blanco & Barboza, 2005; Hayashi et al., 2022). In this study, fungus gnats displayed foraging behavior despite the absence of detectable nectar in the flowers. Volatile analysis via headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME/GC–MS) identified β-pinene (9.55%) and linalool (43.18%) as dominant floral scent components. While β-pinene has been associated with various mimicry systems (Stökl et al., 2011), its prominence as a characteristic odor of decaying wood (Kaiser, 2011) suggests that C. muscicola may exploit this scent to attract adult fungus gnats seeking shelter or foraging sites in forest habitats. Similarly, although linalool is a ubiquitous floral compound (Zhang et al., 2023), its significance as a major volatile in certain mushrooms (e.g., Mycena; Han et al., 2022) may support the hypothesis that it could function in olfactory mimicry of fungal brood sites to attract egg-laying females. This potential mimicry is plausible because females of many species in this family habitually oviposit on fungi, with their larvae feeding on hyphae (Shen et al., 2009). In Yunnan, Sciaridae species are major pests of edible fungi, with those from the genera Bradysia and Lycoriella being the predominant ones (Shen et al., 2009). Although no eggs were observed on the flowers, the exclusive capture of female pollinators may support the hypothesis that C. muscicola employs a dual deception strategy, simultaneously imitating both foraging substrates and oviposition sites to attract fungus gnats. In orchids, inconspicuous floral displays, often accompanied by specific scent profiles, are frequently linked to pollination via sexual deception or brood-site mimicry (Ayasse et al., 2011; Vereecken & Schiestl, 2009). From a pollination syndrome perspective (Fenster et al., 2004), numerous species exhibiting analogous floral morphology likely employ fungus gnat pollination as well. Supporting this view, we documented that the Sciaridae pollination system extends beyond C. muscicola to include another inconspicuous epiphytic orchid, Bulbophyllum shweliense (Bulbophyllinae), which also flowers during the rainy season (June) in the LBZ ancient tea garden (Appendix S1: Figure S1a). This was confirmed through observations of 30 flowers over a 3-h period on 26 June 2023, during which all four recorded female gnats entered the floral channel, became temporarily trapped, and upon escaping after a struggle of tens of seconds (Appendix S1: Figure S1b), had pollinia adhering to their dorsal surfaces (Appendix S1: Figure S1c), thereby demonstrating their effectiveness as pollinators. Although these data are preliminary, they suggest a potentially convergent pollination system. Based on floral traits and visitor morphology, C. muscicola and B. shweliense are likely visited by different gnat taxa, possibly from distinct genera. Future studies incorporating DNA barcoding and floral scent analyses for B. shweliense would therefore offer valuable insights into the convergent evolution of brood-site mimicry systems. To our knowledge, this study documents previously unreported fungus gnat pollination systems in both the Eriinae and Bulbophyllinae subtribes of Orchidaceae. This discovery expands the known pollination strategies between orchids and Sciaridae fungus gnats beyond sexual deception to include brood site mimicry and feeding site mimicry. We have recorded at least two inconspicuously colored, small-flowered orchid species being pollinated by Sciaridae fungus gnats. In orchids, such inconspicuously colored small flowers are primarily found within several genera of the Epidendroideae subfamily. For instance, the Pleurothallidinae tribe (over 5500 species) includes the genus Pleurothallis, the Bulbophyllinae tribe (over 2100 species) includes the genus Bulbophyllum, and the Malaxidinae tribe (over 1200 species) includes the genera Crepidium, Liparis, and Oberonia (Ackerman et al., 2023). The pollination relationships between these orchids and fungus gnats may have been largely overlooked due to the inconspicuous nature of both the flowers and their pollinators. As a result, the pollination relationships between orchids and micro-dipteran insects may be far more diverse than currently recognized. Understanding the interactions between orchids and unassuming fungus gnats (e.g., Mycetophilidae, Sciaridae, Cecidomyiidae) is therefore critical for deciphering the evolution of orchid pollination syndromes and plant–insect co-adaptation. Jiang-Yun Gao and Shi-Mao Wu designed the experiment, and wrote and reviewed the manuscript; Shi-Mao Wu and Sheng Zhang conducted field surveys and data collection; Shi-Mao Wu and Jiang-Yun Gao performed the analysis and prepared figures and tables. All authors contributed to the article and approved the submitted version. This work was supported by the Joint Special Project on Construction of “First-class Universities and Disciplines” of Yunnan University (202201BF070001-017). The authors declare no conflicts of interest. Data (Wu, 2025) are available in Figshare at https://doi.org/10.6084/m9.figshare.29117735.v3. 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